Tim Bushnell, PhD
Tim Bushnell, PhD

Tim Bushnell holds a PhD in Biology from the Rensselaer Polytechnic Institute. He is a co-founder of—and didactic mind behind—ExCyte, the world’s leading flow cytometry training company, which organization boasts a veritable library of in-the-lab resources on sequencing, microscopy, and related topics in the life sciences.

Articles Written By Tim Bushnell, PhD

How To Buy A Flow Cytometer - What You Need To Evaluate From A To Z

By: Tim Bushnell, PhD

So you have the money to buy a flow cytometer. Is it a sorter? Or perhaps a spectral analyzer? No wait, maybe an imaging mass cytometer?  Big or small?  What to choose?  How to choose?  More importantly, once you sign the contract to purchase the instrument, you don’t want to be struck with buyers remorse.  It is indeed a big decision and we have the best advice for you to consider before making the purchase. Let’s discuss some of the steps you should take to prevent buyers remorse and ensure you are getting the best instrument for your needs.  Do…

How small can you go? Flow cytometry of bacteria and viruses

By: Tim Bushnell, PhD

Flow cytometers are traditionally designed for measuring particles, like beads and cells. These tend to fall in the small micron size range. Looking at the relative size of different targets of biological interest, it is clear the most common targets for flow cytometry (cells) are comparatively large (figure 1). Figure 1:  Relative size of different biological targets of interest. Image modified from Bioninja.    In the visible spectrum, where most of the excitation light sources reside, it is clear the cells are larger than the light. This is important as one of the characteristics that we typically measure is the amount…

What Is Spectral Unmixing And Why It's Important In Flow Cytometry

By: Tim Bushnell, PhD

As the labeled cell passes through the interrogation point, it is illuminated by the excitation lasers. The fluorochromes, fluoresce; emitting photons of a higher wavelength than the excitation source. This is typically modeled using spectral viewers such as in the figure below, which shows the excitation (dashed lines) and emission (filled curves) for Brilliant Violet 421TM (purple) and Alexa Fluor 488Ⓡ (green).  Figure 1: Excitation and emission profiles of BV421TM and AF488Ⓡ  In traditional fluorescent flow cytometry (TFF), the instrument measures each fluorochrome off an individual detector. Since the detectors we use — photomultiplier tubes (PMT) and avalanche photodiodes (APD)…

How To Extract Cells From Tissues Using Laser Capture Microscopy

By: Tim Bushnell, PhD

Extracting specific cells still remains an important aspect of several emerging genomic techniques. Prior knowledge about the input cells helps to put the downstream results in context. The most common isolation technique is cell sorting, but it requires a single cell suspension and eliminates any spatial information about the microenvironment. Spatial transcriptomics is an emerging technique that can address some of these issues, but that is a topic for another blog.  So what does a researcher who needs to isolate a specific type of cell do? The answer lies in the technique of laser capture microdissection (LCM). Developed at the National…

The Importance Of Quality Control And Quality Assurance In Flow Cytometry (Part 4 Of 6)

By: Tim Bushnell, PhD

Incorporating quality control as a part of the optimization process in  your flow cytometry protocol is important. Take a step back and consider how to build quality control tracking into the experimental protocol.  When researchers hear about quality control, they immediately shift their attention to those operating and maintaining the instrument, as if the whole weight of QC should fall on their shoulders.   It is true that core facilities work hard to provide high-quality instruments and monitor performance over time so that the researchers can enjoy uniformity in their experiments. That, however, is just one level of QC.  As the experimental…

How To Optimize Instrument Voltage For Flow Cytometry Experiments (Part 3 Of 6)

By: Tim Bushnell, PhD

As we continue to explore the steps involved in optimizing a flow cytometry experiment, we turn our attention to the detectors and optimizing sensitivity: instrument voltage optimization.  This is important as we want to ensure that we can make as sensitive a measurement as possible.  This requires us to know the optimal sensitivity of our instrument, and how our stained cells are resolved based on that voltage.  Let’s start by asking the question what makes a good voltage?  Joe Trotter, from the BD Biosciences Advanced Technology Group, once suggested the following:  Electronic noise effects resolution sensitivity   A good minimal PMT…

Optimizing Flow Cytometry Experiments - Part 2 How To Block Samples (Sample Blocking)

By: Tim Bushnell, PhD

In my previous blog on  experimental optimization, we discussed the idea of identifying the best antibody concentration for staining the cells. We did this through a process called titration, which  focuses on finding the best signal-to-noise ratio at the lowest antibody concentration. In this blog we will deal with sample blocking As a reminder, there are two other major binding concerns with antibodies. The first is the specific binding of the Fc fragment of the antibody to the Fc Receptor expressed on some cells. This protein is critical for the process of destroying microbes or other cells that have been…

How To Determine The Optimal Antibody Concentration For Your Flow Cytometry Experiment (Part 1 of 6)

By: Tim Bushnell, PhD

Over the next series of blog posts, we will explore the different aspects of optimizing a polychromatic flow cytometry panel. These steps range from figuring out the best voltage to use, which controls are critical for data interpretation, what quality control tools can be integrated into the assay; how to block cells, and more. This blog will focus on determining the optimal antibody concentration.  As a reminder about the antibody structure, a schematic of an antibody is shown below.  Figure 1: Schematic of an antibody. Figure from Wikipedia. The antibody is composed of two heavy chains and two light chains that…

2020 - A Year Turned Upside Down

By: Tim Bushnell, PhD

What an incredible year 2020 has been. It started off like any other year and bam SARS-CoV-2 (aka COVID 19) entered the equation, bringing chaos and havoc to the world. Things kept changing overnight as new rules and regulations popped up. Masking, quarantine, and flatten the curve became common words in the news. How we met, how we interacted changed almost overnight. Throughout all of this, as we look to 2021, there is hope and optimism. Multiple vaccines have been developed, building on years of research into the SARS-CoV virus, with some approved for human use, and others on the horizon.…

Brightness Is In The Eye Of The Detector - What To Consider When Designing Your Panel

By: Tim Bushnell, PhD

The heart and soul of the flow cytometry experiment is the ‘panel.’ The unique combinations of antibodies, antigens, fluorochromes, and other reagents are central to identifying the cells of interest and extracting the data necessary to answer the question at hand. Designing the right panel for flow cytometry is essential for detecting different modalities. The more parameters that can be interrogated will yield more information about the target cells. Current instruments can measure as many as 40 different parameters simultaneously. This is exciting, as it allows for more complex questions to be studied. Panel design is also valuable for precious samples,…

Tools to Improve Your Panel Design – Determining Antigen Density

By: Tim Bushnell, PhD

When a researcher chooses to use flow cytometry to answer a scientific question, they first have to build a polychromatic panel that will take advantage of the power of the technology and experimental design. When we set up to use flow cytometry to answer a scientific question, we have to design a polychromatic panel that will allow us to identify the cells of interest – the target of the research.  To identify these cells, we need to build a panel that takes advantage of the relative brightness of the fluorochromes, the expression level of the different proteins on the cell,…

This Is How Full Spectrum Cytometry Works

By: Tim Bushnell, PhD

There are 4 major ways to sort cells. The first way can use magnetic beads coupled to antibodies and pass the cells through a magnetic field. The labeled cells will stick, and the unlabeled cells will remain in the supernatant. The second way is to use some sort of mechanical force like a flapper or air stream that separates the target cells from the bulk population. The third way is the recently introduced microfluidics sorter, which uses microfluidics channels to isolate the target cells. The last method, which is the most common––based on Fuwyler’s work––is the electrostatic cell sorter. This…